Nucleic acid inhibitors of glutamate receptors

ABSTRACT

The present invention relates to novel nucleic acid ligands or aptamers that bind to and inhibit the activation of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) subtype of ionotropic glutamate receptors. Also disclosed is a novel combination of technologies, i.e., SELEX and laser pulse photolysis for the selection and screening of aptamers that inhibit receptor function and are useful therefore, in the treatment of diseases associated with excessive activation of ionotropic glutamate receptors.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of co-pending U.S. application Ser. No.12/578,231 filed Oct. 13, 2009, which is a divisional of U.S.application Ser. No. 11/256,726 filed on Oct. 24, 2005, which issued asU.S. Pat. No. 7,601,823 on Oct. 13, 2009; and claims priority to U.S.provisional application Ser. No. 60/621,285 filed Oct. 22, 2004; theentire disclosures of each are incorporated herein by reference.

STATEMENT OF RIGHTS UNDER FEDERALLY-SPONSORED RESEARCH

This invention was made with government support under grantW81XWH-04-1-0106 awarded by the U.S. Department of Defense and grantGM60411 awarded by the National Institutes of Health. The government hascertain rights in the invention.

FIELD OF THE INVENTION

The invention relates to methods for the identification and preparationof nucleic acid ligands to AMPA glutamate receptors. Included in theinvention are specific RNA ligands to AMPA glutamate receptors whichinhibit the activity of these receptors.

BACKGROUND OF THE INVENTION

Ion channel glutamate receptors are ligand-gated transmembrane proteinsthat can be activated by the binding of glutamate, the principalexcitatory neurotransmitter in the brain. Ionotropic glutamate receptors(iGluRs) are, therefore, the major excitatory neurotransmitter receptorproteins in the mammalian brain. As such, these receptors play specialroles in brain activities, such as memory and learning, and have beenimplicated in a variety of neurological diseases, such as post-strokecellular lesion and amyotrophic lateral sclerosis [Dingledine et al.,1999; Heath and Shaw 2002].

When glutamate, released from a presynaptic neuron, binds to apostsynaptic glutamate receptor, the receptor rapidly changes itsconformation and transiently forms an open ion channel, thus resultingin a change of the postsynaptic membrane potential. A postsynapticpotential of sufficient strength triggers an action potential, whichwill in turn propagate the initial nerve impulse. The major function ofiGluRs is to mediate fast synaptic neurotransmission underlying thebasic activities of the brain, for example, memory and learning.Excessive activation of ionotropic glutamate receptors, particularly theα-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) subtype, isknown to induce calcium-dependent excitotoxicity. Excitotoxicity hasbeen considered as a general mechanism underlying a number ofneurodegenerative disorders such as amyotrophic lateral sclerosis (ALS),stroke, Alzheimer's disease and Parkinson's syndrome.

Using inhibitors to dampen the excessive activity of these receptors mayserve as a treatment for neurological disorders such as amyotrophiclateral sclerosis (ALS) or Huntington's disease. To date, Riluzole, aninhibitor of presynaptic glutamate release, is the only drug to producea significant benefit to the survival of ALS patients. The number ofglutamate receptor inhibitors currently available is limited and theseinhibitors generally show cross activity to other receptors, forexample, kainate receptors. The cross activity is not desirable, becausethe AMPA and kainate receptors have functional differences. Furthermore,the majority of AMPA receptor inhibitors have poor water solubility. Inaddition, there is a lack of an assay of inhibitor-receptor interactionswithin the microsecond (μs) to millisecond (ms) time domain. This isbecause an AMPA receptor opens its channel in the μs time scale anddesensitizes within a few ms in the continued presence of glutamate.Consequently, the affinity of all AMPA receptor inhibitors has beendetermined only with the desensitized receptors. These deficiencies havesignificantly hampered drug development.

What is needed, therefore, is an AMPA glutamate receptor inhibitor thatis characterized by a high affinity for its target, preferably in thenanomolar range, specificity targeting the glutamate receptor, excellentwater solubility and relevance of its inhibitory properties to thefunctional forms of the receptor rather than the desensitized receptorforms.

SUMMARY OF THE INVENTION

The present invention provides a new class of water-soluble highaffinity compounds, specifically nucleic acid ligands or aptamers thatinhibit glutamate binding to the AMPA glutamate receptor.

In one aspect, therefore, the invention relates to novel nucleic acidligands to AMPA receptors. The nucleic acid ligands or aptamers of theinvention are selected by an in vitro iterative process of selection,partitioning and amplification referred to as SELEX. Additionally, thenucleotides of the aptamer may be chemically modified either prior to orafter selection of the aptamers by SELEX. In one embodiment, the nucleicacid of the invention is an RNA. Examples of glutamate inhibitors of theinvention are nucleic acids that have a nucleotide sequence selectedfrom the group consisting of the sequences set forth in SEQ ID NO.: 1 toSEQ ID NO.: 11.

In a related aspect, the present invention relates to a method ofidentifying a nucleic acid ligand that can inhibit glutamate receptorfunction, the method comprising the steps of screening a nucleic acidlibrary for a nucleic acid that binds to a glutamate receptor (SELEX);providing a cell that has been transfected to overexpress the glutamatereceptor; exposing the cell to glutamate in the presence and absence ofthe nucleic acid identified by the SELEX method and measuring theglutamate-induced whole-cell current using laser pulse photolysis ofcaged glutamate as a source of glutamate. The measurements forwhole-cell current in the absence and presence of the nucleic acid arecompared. A decrease in whole-cell current in the presence of thenucleic acid indicates that the nucleic acid is a specific glutamatereceptor inhibitor.

In another aspect, the invention relates to a method of modulating thefunction of a glutamate receptor comprising contacting a receptor with anucleic acid ligand that specifically binds the receptor and has beenshown to have an inhibitory effect.

In yet another aspect, the invention relates to a pharmaceuticalcomposition comprising the nucleic acids of the invention andoptionally, a pharmaceutically acceptable carrier.

In a related aspect, the invention relates to a method of treatment fora disease or condition characterized by excessive activation ofionotropic glutamate receptors, the method comprising administering tothe subject in need of such treatment, a therapeutically effectiveamount of the nucleic acid of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide sequences (SEQ ID NOs: 1-6 and 8-11) ofnucleic acid ligands of the invention.

FIG. 2(A-D) shows the results of the determination of the specificinhibition of the GluR2 AMPA receptor by the selected aptamer, AN57 (SEQID NO. 6) using whole-cell recording of HEK-293 cell expressing thereceptor channel.

FIG. 3(A-B) shows the characterization of the inhibitory properties ofone embodiment of the invention, aptamer AN57 with the GluR2Q_(flip)AMPA receptor channel. As shown, AN57 is a competitive inhibitor.

FIG. 4(A-D) shows a proposed mechanism of action of aptamer AN57, which,in one embodiment assumes two structurally distinct species, both ofwhich are required for inhibition.

FIG. 5(A-B) shows that aptamer AN57 has inhibitory properties rivalingthose of a known, very potent glutamate receptor inhibitor, NBQX.

FIG. 6 is a graph that shows the dependence of the whole-cell currentresponse of the GluR2Q_(flip) receptor channel to glutamate in theabsence (upper curve) and presence (lower curve) of 5 mM NBQX. Thisshows NBQX is a competitive inhibitor.

FIG. 7 shows that when purified, aptamer AN57 assumes two structures (M1and M2) either of which individually does not inhibit the whole-cellresponse to kainate (kainate was used to induce the GluR2Q_(flip) AMPAreceptor response). Inhibition seen with unpurified AN57 is restoredwhen the M1 and M2 are recombined.

FIG. 8 shows that M1 and M2 individually do not inhibit the whole-cellresponse to glutamate via GluR2Q_(flip) receptor channel. As withkainite, inhibition seen with unpurified AN57 is restored when the M1and M2 are recombined.

DETAILED DESCRIPTION OF THE INVENTION

All patents, publications, applications and other references citedherein are hereby incorporated by reference into the presentapplication. In the description that follows, certain conventions willbe followed as regards the usage of terminology.

The term “aptamer” refers to a nucleic acid or oligonucleotide moleculethat binds to a specific molecular target such as a protein receptor.Aptamers are obtained from an in vitro evolutionary process known asSELEX (Systematic Evolution of Ligands by EXponential Enrichment), whichselects target-specific aptamer sequences from large combinatoriallibraries of single stranded oligonucleotide templates comprisingrandomized sequences. Aptamer compositions may be double-stranded orsingle-stranded, and may include deoxyribonucleotides, ribonucleotides,nucleotide derivatives, or other nucleotide-like molecules. Thenucleotide components of an aptamer may include modified or non-naturalnucleotides, for example nucleotides that have modified sugar groups(e.g., the 2′-OH group of a ribonucleotide may be replaced by 2′-F or2′-NH.sub.2). which may improve a desired property, e.g., resistance tonucleases or longer lifetime in blood.

In a preparation of nucleic acids of the invention, individual aptamershaving the same nucleotide sequence may differ in their secondarystructure. Aptamers may also be conjugated to other molecules, e.g., ahigh molecular weight carrier to slow clearance of the aptamer from thecirculatory system. Aptamers may be specifically cross-linked to theircognate ligands, e.g., by photo-activation of a cross-linker. [Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-13].

The term “nucleic acid” means either DNA, RNA, single-stranded ordouble-stranded and any chemical modifications thereof. Modificationsinclude, but are not limited to, those which provide other chemicalgroups that incorporate additional charge, polarizability, hydrogenbonding, electrostatic interaction, and fluxionality to the nucleic acidligand bases or to the nucleic acid ligand as a whole. Suchmodifications include, but are not limited to, 2′-position sugarmodifications, 5-position pyrimidine modifications, 8-position purinemodifications, modifications at exocyclic amines, substitution of4-thiouridine, substitution of 5-bromo or 5-iodo-uracil, backbonemodifications, methylations, unusual base-pairing combinations such asthe isobases isocytidine and isoguanidine and the like. Modificationscan also include 3′ and 5′ modifications such as capping.

The present invention provides novel nucleic acids that inhibit theactivity of ionotropic glutamate receptors, and in particular, theα-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) subtype ofglutamate receptors. The nucleotide sequence of examples of nucleicacids of the invention are shown in FIG. 1.

The present invention further provides a novel method of identifyingnucleic acids which specifically bind to and inhibit the function ofglutamate receptors such as the AMPA receptor. The method comprisesfirst screening a nucleic acid library for a nucleic acid that binds toa glutamate receptor. A modified SELEX method was used to identify thenucleic acid ligands disclosed herein. Once a glutamatereceptor-specific aptamer has been identified, the aptamer's ability toinhibit glutamate function is evaluated. By providing a cell that hasbeen transfected to overexpress the glutamate receptor and measuringglutamate-induced whole-cell current in a single cell in the presenceand absence of the nucleic acid identified by SELEX, a comparison of themeasurement of whole cell current in the presence and absence of nucleicacid is informative of the aptamer's potential as a glutamate receptorinhibitor. A decrease in the whole-cell current in the cell in thepresence of nucleic acid as compared to the whole-cell current in theabsence of nucleic acid indicates that the nucleic acid inhibitsglutamate receptor function.

To arrive at the novel inhibitors of the present invention, therefore, anovel combination of two approaches was used, namely an in vitroiterative procedure, SELEX, to select the aptamers of the invention froma combinatorial RNA library and a laser-pulse photolysis technique thathas a microsecond (μs) time resolution to screen the aptamers against afunctional (i.e., non-desensitized) form of the glutamate receptor.

Aptamers that recognize the AMPA receptors may be selected in a numberof ways. In one embodiment, aptamers are selected from a combinatoriallibrary using SELEX, by immobilizing intact cells containing theglutamate receptor on a biosensor chip and monitoring using a surfaceplasmon resonance (SPR) technique. SPR is an optical technique thatoffers real time analysis of the rates of adsorption and desorption fora range of surface interactions. In an alternate embodiment, a cellmembrane preparation, for example from a cell that has been transfectedto overexpress the target receptor, may be used as the selection target.

Known inhibitors for the receptor, for example, NBQX, philanthotoxin-343and GYM 47261 are used to displace all specific RNAs previously bound tothe receptor. The consensus sequences in the aptamers are thenidentified by cloning and sequencing to identify inhibitor candidates.

Prior to screening the aptamers for inhibitory activity, the targetreceptor subunits are expressed at an enhanced efficiency bytransfecting cells, for example, HEK-293 cells with a nucleic acidencoding the receptor subunit(s) in conjunction with a nucleic acidencoding simian virus (SV) 40 T antigen. Methods for the construction ofan appropriate vector and for transfection of an appropriate host cellare well known to those of skill in the art and are described, forexample, in Sambrook et al., 2001, Molecular Cloning: A LaboratoryManual, Third Edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; DNA Cloning: A Practical Approach, Volumes I and II, 1985(D. N. Glover ed.); Oligonucleotide Synthesis, 1984 (M. L. Gait ed.)

To elucidate the channel opening kinetics of the homomeric channel withand without aptamer ligand, a rapid kinetic technique that has amicrosecond time resolution, namely laser pulse photolysis of cagedglutamate, is used. The channel opening rate constant, the channelclosing rate constant or the lifetime of the channel, and thedissociation equilibrium constant for glutamate are determined. Thus, bythis methodology, the mechanism of action, the affinity, and selectivityof each aptamer on the functional forms of each receptor subunit can becharacterized.

Each of the steps of the method for identifying nucleic acid ligandsthat may useful as glutamate receptor inhibitors are discussed in detailbelow.

Aptamer Selection by SELEX

A method for the in vitro evolution of nucleic acid molecules with highaffinity binding to target molecules is known to those of skill in theart and is described in U.S. Pat. No. 5,270,163. The method, known asSELEX (Selective Evolution of Ligands by EXponential Enrichment)involves selection from a mixture of candidate oligonucleotides from alibrary comprising a large sequence variations (in the present case,˜10¹⁵) and step-wise iterations of binding, partitioning andamplification, using the same general selection theme, to achievevirtually any desired criterion of binding affinity and selectivity.

Starting from a mixture of nucleic acids, preferably comprising asegment of randomized sequence, the SELEX method includes the steps ofcontacting the mixture with the desired target, partitioning unboundnucleic acids from those nucleic acids which have bound to the targetmolecule, dissociating the nucleic acid-target complexes, amplifying thenucleic acids dissociated from the nucleic acid-target complexes toyield a ligand-enriched mixture of nucleic acids, then reiterating thesteps of binding, partitioning, dissociating and amplifying through asmany cycles as desired to yield high affinity nucleic acid ligands tothe target molecule.

The SELEX method may be modified to encompass the identification ofhigh-affinity nucleic acid ligands containing modified nucleotidesconferring improved characteristics on the ligand, such as improved invivo stability or improved delivery characteristics. Examples of suchmodifications include chemical substitutions at the ribose and/orphosphate and/or base positions. SELEX-identified nucleic acid ligandscontaining modified nucleotides are described, for example, in U.S. Pat.No. 5,660,985. These include oligonucleotides containing nucleotidederivatives chemically modified at the 2′ position of ribose, 5 positionof pyrimidines, and 8 position of purines. U.S. Pat. No. 5,756,703describes oligonucleotides containing various 2′-modified pyrimidines.U.S. Pat. No. 5,580,737 describes highly specific nucleic acid ligandscontaining one or more nucleotides modified with 2′-amino (2′-NH.sub.2),2′-fluoro (2′-F), and/or 2′-O-methyl (2′-OMe) substituents.

Modifications of the nucleic acid ligands contemplated in this inventioninclude, but are not limited to, those which provide other chemicalgroups that incorporate additional charge, polarizability,hydrophobicity, hydrogen bonding, electrostatic interaction, andfluxionality to the nucleic acid ligand bases or to the nucleic acidligand as a whole. Such modifications include, but are not limited to,2′-position sugar modifications, 5-position pyrimidine modifications,8-position purine modifications, modifications at exocyclic amines,substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil;backbone modifications, phosphorothioate or alkyl phosphatemodifications, methylations, unusual base-pairing combinations such asthe isobases isocytidine and isoguanidine and the like. Modificationscan also include 3′ and 5′ modifications such as capping.

The modifications can be pre- or post-SELEX process modifications.Pre-SELEX process modifications yield nucleic acid ligands with bothspecificity for their SELEX target and improved in vivo stability.Post-SELEX process modifications made to 2′-OH nucleic acid ligands canresult in improved in vivo stability without adversely affecting thebinding capacity of the nucleic acid ligand.

In one embodiment, the selection of a nucleic acid ligand specific forthe target of interest may be carried out on a sensor chip surface andmonitored by SPR. A combinatorial RNA library is made by transcribingDNA templates or a DNA library. Each template consists of 108 bases witha 40-base randomized segment. This segment is flanked by two constantregions for primer annealing. The 5′ constant sequence includes apromoter for T7 RNA polymerase. The two restriction sites are EcoRIlocated in the 5′ constant region and HindIII located in the 3′ constantregion, respectively.

Rat motor neurons contain all AMPA receptor subunits and may be used asthe protein target in SELEX. The motor neurons are dissected from thespinal cord of 15-day old embryonic rats, and purified by “panning” thecells in a Petri dish pre-coated with an anti-mouse IgG, 192-IgG, whichrecognizes the p75 neurotrophic factors that are solely expressed in the15-day-old motor neurons.

The binding of RNAs to AMPA receptors (step 1) is carried out on abiosensor chip surface and monitored using the SPR technique in realtime. In one embodiment, 192-IgG is covalently immobilized on a CMSsensor chip using a standard carbodiimide reagent. Then the motorneurons are tethered onto the sensor chip surface by binding to theantibody. The RNA molecules are injected into the reaction cell. Thosethat can bind to the AMPA receptors on the motor neurons immobilized onthe surface will be retained. The RNA selection reaction is carried outat 22° C. in the presence of anti-RNAse. One of the advantages of thissensor technique is that as low as pictogram quantity of the protein issufficient for selection. The binding of RNAs register as a net increaseof SPR signal, while the subsequent elution of the bound RNAs in thepresence of known inhibitors results in a fall of the SPR signal. Bothprocesses are monitored in real time.

A mixture of known inhibitors is injected into the reaction cell (step2). The RNA molecules bound to the motor neurons at the same and/ormutually exclusive sites will be eluted all at once, and collected. Forpurposes of practicing the present invention, inhibitors include, NBQX(a competitive inhibitor), philanthotoxin-343, a wasp neurotoxin analog(non-competitive inhibitor) and GYM 47261, a 2,3-benzodiazepinederivative. The collected RNAs are reverse-transcribed and amplified byPCR (step 3). The new RNA pool is prepared by transcription. A new roundof selection can then proceed from step 1. The SELEX progress ismonitored for the level of SPR signal generated by the binding of theRNAs from a certain round, for instance, the selection is completed whenthe maximum SPR signal between two consecutive rounds no longer changes.When SELEX is completed (usually 8-12 rounds), the cDNA pool is cut withEcoRI and HindIII at the constant regions and ligated into the vectorpGEM-3Z. Colonies are sequenced and the consensus sequences identified.

In a preferred embodiment, using an improvement to SELEX technology,aptamers against a membrane-bound receptor were identified using, as thetarget, cell membranes from fragmented HEK-293 cells that had beentransfected to overexpress the receptor. The RNA library was describedpreviously (Fan, X., Shi, H., Adelman, K. & L is, J. T. Probing TBPinteractions in transcription initiation and reinitiation with RNAaptamers that act in distinct modes. Proc Natl Acad Sci USA 101,6934-9). The in vitro transcribed RNA was dissolved in extracellularbuffer (see buffer composition below) at a final concentration of 20 μM.The RNA library was heated at 70° C. for 10 min before use. Thefragmented HEK-293 cell membrane was prepared in accordance with methodsknown to those of skill, and the membrane-bound receptor was adjusted toa final concentration of 50 nM, as determined by [³H]AMPA binding. Thereaction mixture was incubated at 22° C. for 40 min for RNA binding inthe presence of 0.3 units/μl RNase inhibitor (Ambion, Austin, Tex.). Inan improvement of the SELEX technology, reaction mixture containing thenucleic acid-target complexes was passed through a nitrocellulose filterusing a filtration apparatus (Pierce, Rockford, Ill.) to wash offunbound RNAs. The desired RNAs were eluted using 1 mM-NBQX and were thensubjected to reverse transcription and PCR. A new RNA pool was thentranscribed, and a new round of selection was repeated. Overall, 15rounds of SELEX were performed, including 3 negative selections usingHEK-293 cell membrane that did not express any AMPA receptor. About 120clones from rounds 12, 14 and 15, were sequenced. The clones harboringconsensus sequences were identified and assayed for inhibition.

In order to establish a baseline for the characterization of theefficacy and selectivity of each aptamer, the channel opening kineticsof each of the AMPA receptor subunits was determined using themeasurement of glutamate-induced whole cell current in AMPAreceptor-bearing cells. To improve the current response of a singlecell, however, it is desirable to enhance the expression of the receptorof which the activity is to be measured. A method for enhancing proteinexpression in single HEK 293 is summarized below (Huang et al., Journalof Neruoscience Methods, 142:159-166 2005.)

Transfection for Enhanced Expression of GluR2Q_(flip)

Recombinant proteins are routinely expressed in heterologous expressionsystems, such as human embryonic kidney 293 (HEK 293) cells. Theefficiency of the expression is critical when the expressed protein mustbe characterized at the single-cell level. A simple method by which theprotein expression efficiency in single HEK 293 cells is enhanced bycoexpressing simian virus 40 large T antigen (TAg), a powerfuloncoprotein. Using this technique, GluR2 ionotropic glutamate receptorexpression in single HEK 293S cells increased approximately seven-fold.The ratio of the plasmid amount of TAg to that of the receptor wasoptimized at 1:10, while the receptor function was unaffected in thepresence of TAg. Fluorescence imaging of a population of cells was usedas an independent detection method and a similar increase in expressionof green fluorescent protein (GFP) by TAg coexpression was found. Thus,this method is suitable for enhancing the expression of both membraneand soluble proteins at the single-cell level for purposes of practicingthe present invention. Additionally, the function of a protein can bestudied directly in intact cells, a feature particularly useful forstudying membrane proteins.

Enhance expression efficiency is achieved by coexpression of simianvirus (SV) 40 large T antigen (TAg), a powerful oncoprotein (Ali andDeCaprio, 2001; Chen and Hahn, 2003; Simmons, 2000; Sullivan and Pipas,2002), with the protein of interest. Specifically, a nucleic acidencoding the protein of interest is harbored in a plasmid containing theSV40 replication origin, and the TAg gene is encoded in a separatevector. Transient coexpression of TAg produces more proteins of interestper cell. Specifically, TAg enhancement of the single-cell expression ofGluR2, a key glutamate receptor subunit (Li et al., 2003b), wascharacterized to establish the optimal plasmid ratio and the mostcomplementing cell line. The function of the GluR2 receptor withinintact cells was further characterized without removing TAg. Inaddition, using green fluorescence protein (GFP) (Chalfie et al., 1994)as a reporter gene and fluorescence imaging of a population of HEK 293cells as an independent detection method, it was shown that GFPexpression in these cells increased similarly.

Therefore, a plasmid encoding each of the AMPA receptor subunits,GluR1-4, and the GluR6Q kainate receptor subunit was used to transientlyexpress the corresponding homomeric channel in HEK-2935 cells by astandard calcium phosphate method. As a cell marker forelectrophysiological recording but not for SELEX, the cell wascotransfected with the plasmid encoding green fluorescent protein (GFP).The ratio by weight of the plasmid of GFP to that of a receptor was 1:10and the receptor plasmid used was ˜3-10 μg/35 mm Petri dish. The SV40large T-antigen (TAg) gene was also cotransfected to enhance thesingle-cell receptor expression, as we previously reported. The ratio ofthe plasmid by weight for TAg to that of a receptor was 1:10. The cellswere maintained in Dulbecco's modified Eagle's medium supplemented with10% fetal bovine serum (all from Invitrogen, Carlsbad, Calif.), in a 37°C., 10% CO₂, humidified incubator. 48 hours after transfection, thecells were either used for patch clamping or harvested for SELEX.

Exploiting the enhanced expression of the AMPA glutamate receptor intransfected cells, the ability of the nucleic acid aptamers of theinvention to impact glutamate receptor function was evaluated.

Whole-Cell Current Measurement

Glutamate, a natural neurotransmitter, was used as the activatingligand, and the resulting receptor response in a single cell wasrecorded. Recording electrodes were made from glass capillary pipettesand fire polished. The electrode resistance was ˜3MΩ when filled withthe electrode solution. The electrode solution contained 110 mM CsF, 30mM CsCl, 4 mM NaCl, 0.5 mM CaCl₂, 5 mM EGTA, and 10 mM Hepes (pH 7.4adjusted by CsOH). The external bath solution contained 150 mM NaCl, 3mM KCl, 1 mM CaCl₂, 1 mM MgCl₂ and 10 mM Hepes (pH7.4 adjusted by NaOH).The GFP fluorescence in transfected cells was visualized using anAxiovert S100 microscope with a fluorescent detection system from CarlZeiss (Thornwood, N.Y.). The whole-cell current was recorded using anAxonpatch-200B amplifier at a cutoff frequency of 2-20 kHz by a built-ineight-pole Bessel filter and digitized at 5-50 kHz sampling frequencyusing a Digidata 1322A from Axon Instruments (Union City, Calif.). Thedata acquisition software was pCLAMP 8, also from Axon Instruments.

Laser-Pulse Photolysis

Laser pulse photolysis using caged neurotransmitters has been designedto measure the receptor channel opening kinetics and inhibitor-receptorinteraction with a μs time resolution. Caged neurotransmitters arebiologically inert, but photolabile precursors of neurotransmitters.This technique utilizes a rapid photolytic release of a neurotransmitterfrom the caged precursor within the μs time domain to overcome theotherwise slow diffusion and mixing of free neurotransmitters with thereceptor on the surface of a cell. Using this technique, the opening ofa receptor channel can be measured prior to receptor desensitization. Tostudy glutamate receptors, caged glutamate was developed and iscommercially available. The setup for the laser-pulse photolysisexperiment is described elsewhere [Matsubara et al., Biochemistry31:5507-14, 1992; Niu et al., Chemical kinetic investigations ofneurotransmitter receptors on a cell surface in a microsecond timeregion, Vol. VII, Academic Press, New York, 1996]

For purposes of practicing the method of the present invention, αCNBglutamate (caged glutamate) from Molecular Probes (Eugene, Oreg.) wasdissolved in the external bath buffer and delivered over the surface ofa HEK-293 cell expressing a receptor using a U-tube made from stainlesssteel tubing with a hole of ˜150 μm in diameter. The addition of thesolution containing the ligand and the suction of the waste arecontrolled by two peristaltic pumps. A HEK-293 cell is ˜15 μm indiameter. The cell suspended from the current-recording electrode is˜100 μm away from the hole. The linear flow rate of the solution is 1-4cm/s. An optical fiber through which laser light for photolysis isdelivered to the cell has a core diameter of 300 μm. The relativedistance between the cell and the fiber is ˜400 μm.

A U-tube flow device was used to apply glutamate or the caged glutamate(Molecular Probes, Eugene, Oreg.) in the absence and presence of aptamerto a cell expressing the receptor of interest. The current traces weresampled at 5-50 kHz and filtered at 2-20 kHz by an 8-pole Bessel filter.The data were acquired using pCLAMP 8 (Axon Instruments, Union City,Calif.). In the laser-pulse photolysis measurement of thechannel-opening process of the GluR2 AMPA receptor, the caged glutamatedissolved in the external bath buffer was photolyzed to release freeglutamate in the microsecond time region to activate the receptorchannel. A laser pulse from the third harmonic output (355 nm, 8 nspulse length) of a Minilite II pulsed Q-switched Nd:YAG laser(Continuum, Santa Clara, Calif.) was used in the photolysis measurement.All whole-cell recordings were at −60 mV and 22° C.

Once a HEK-293 cell is in the whole-cell mode, it is lifted from thebottom of the dish and suspended in the external bath solution. Afterthe cell is equilibrated with caged glutamate for 250 ms, a laser pulseat 355 nm with a pulse length of 8 ns is generated from a Minilite IIpulsed Q-switched Nd:YAG laser from Continuum (Santa Clara, Calif.)tuned by a third harmonic generator. The laser light was coupled into afiber optic from FiberGuide Industries (Stirling, N.J.) and the powerwas adjusted to be 200-800 μJ, detected by a Joulemeter from Gentec(Quebec, Canada).

The concentration of the photolytically generated glutamate isdetermined by the following method. Known concentrations of freeglutamate are applied to a HEK-293 cell, using the flow device. Thecurrent amplitude obtained is corrected for desensitization using astandard protocol and compared with that obtained from the laser-pulsephotolysis experiment (this is also a means of assaying whetherrepetitive laser pulses have damaged the cell).

In general, a receptor-mediated reaction can be written as follows:

$\begin{matrix}{{A + L}\overset{K_{1}}{\rightleftharpoons}{AL}_{n}\overset{\Phi}{\rightleftharpoons}\begin{matrix}\overset{\_}{{AL}_{n}} \\({open})\end{matrix}} & \; \\{I_{t} = {I_{\max}\left\lbrack {1 - {\exp\left( {{- k_{obs}}t} \right)}} \right\rbrack}} & (1) \\{k_{obs} = {k_{cl} + {k_{op}\left( \frac{L}{L + K_{I}} \right)}^{n}}} & (2)\end{matrix}$

A represents the active form of the receptor, L, the ligandconcentration, AL_(n), the closed-channel forms of the receptor, andAL_(n) the open-channel form, K₁ the intrinsic dissociation constant ofactivating ligand and Φ the channel opening equilibrium constant(Φ=k_(cl)/k_(op)). The k_(cl) is the channel closing rate constant orthe lifetime of the channel, k_(op) is the channel opening rateconstant, and n is the number of ligand binding sites.

Based on this mechanism, the current rises exponentially (Equation 1)due to channel opening. In Equation 1, I_(t) represents the currentobserved at time t, and I_(max) is the maximal observed current in theabsence of receptor desensitization (assuming that the desensitizationoccurs after the channel opening). The relationship between the observedfirst-order rate constant for the channel opening and constants for thegeneral mechanism is shown in Equation 2. The derivation of thisequation assumes that binding of ligand is fast compared to the channelopening process. Plotting the k_(obs) value vs. the concentration, L, ofthe photolytically released glutamate will yield the constants, k_(cl),k_(op), K₁, and n using Equation 2.

Whenever possible, independent approaches are applied to verify some ofthe results obtained from the laser pulse photolysis experiments. Forinstance, k_(cl), the channel closing rate constant, is the same aslifetime of the channel obtained from signal channel recordingtechnique. In laser pulse photolysis experiments, a concentration of thephotolytically released ligand as high as 400 μM has been obtainedwithout damaging the cell. Obtaining a higher concentration is neitherpractical nor necessary. A higher concentration release requires ahigher laser power, which will damage or even destroy the cell.Furthermore, a ligand concentration at 400 μM or even less is generallysufficient to apply the described method to the determination of thereceptor channel opening kinetics.

The mechanism of inhibition of each AMPA receptor channel by theselected aptamers is investigated and the affinity of an aptamer forboth the closed and open forms is measured. The selectivity of anaptamer for all the four AMPA receptor subunits is also determined.

The mechanism of inhibition can be determined by studying the effect ofan inhibitor on the channel opening rate process. For instance, aninhibitor can bind only to the closed channel forms (Mechanism 1: openchannel blockade) or it binds to both the closed and open channel forms(Mechanism 2: noncompetitive mechanism). The observed rate constant,k_(obs), in the presence and absence of inhibitor can be measured usingEquation 1 (in Aim II). The relationship of the k_(obs) to the molarconcentration of the ligand (glutamate), L, and the inhibitor, I, can bewritten according to the individual mechanism; for example, for openchannel blockade (mechanism 1), Equation 3 can be used:

$\begin{matrix}{k_{obs} = {{k_{op}\left( \frac{L}{L + K_{I}} \right)}^{n} + {k_{cl}\left( \frac{{\overset{\_}{K}}_{I}}{{\overset{\_}{K}}_{I} + I} \right)}}} & (3)\end{matrix}$

For the noncompetitive mechanism (2), the following equation (4) wouldbe used:

$\begin{matrix}{k_{obs} = {{{k_{op}\left( \frac{L}{L + K_{I}} \right)}^{n}\left( \frac{{\overset{\_}{K}}_{I}}{{\overset{\_}{K}}_{I} + I} \right)} + {k_{cl}\left( \frac{{\overset{\_}{K}}_{I}}{{\overset{\_}{K}}_{I} + I} \right)}}} & (4)\end{matrix}$

In deriving these equations, one binding site for inhibit per receptormolecule is assumed for simplicity. At low concentrations of glutamate(L<<K₁), k_(obs) reflects the channel closing rate constant since thecontribution of the k_(op) portion in Equations 2, 3 and 4 to theoverall rate, k_(obs), is minimal. Under this condition, the effect ofthe inhibitor on the k_(cl) can be measured. In this case, for bothMechanisms 1 and 2, the effect of the inhibitor on the k_(cl) are thesame and can be obtained by Equation 5, which can be derived from eitherEquation 3 or 4. From Equation 5, the dissociation constant of theinhibitor from the open channel form, K₁, can be uniquely determined.

The effect of the inhibitor on k_(op) is obtained at high ligand(glutamate) concentrations (where k_(obs) is >>k_(cl)). For Mechanism 1,the inhibitor does not affect k_(op) (Equation 6). For Mechanism 2, theinhibitor affects both k_(cl) and k_(op) (Equation 7). Thus, based uponthe effects of an inhibitor on both k_(cl) and k_(op), the mechanism ofaction of an inhibitor can be differentiated. Furthermore, thedissociation constant of the inhibitor for the closed channel form K_(I)can be uniquely determined for Mechanism 2, the noncompetitiveinhibition. In summary, a complete understanding of the mechanism ofaction of a inhibitor cannot be achieved without the knowledge of theeffects of the inhibitor on both k_(cl) and k_(op). Experimentally, thetime resolution provided by the laser pulse photolysis technique makesit possible to measure these effects.

$\begin{matrix}{k_{obs} = {k_{cl}\left( \frac{{\overset{\_}{K}}_{I}}{{\overset{\_}{K}}_{I} + I} \right)}} & (5) \\{{k_{obs} - {k_{cl}\left( \frac{{\overset{\_}{K}}_{I}}{{\overset{\_}{K}}_{I} + I} \right)}} = {k_{op}\left( \frac{L}{L + K_{I}} \right)}} & (6) \\{{k_{obs} - {k_{cl}\left( \frac{{\overset{\_}{K}}_{I}}{{\overset{\_}{K}}_{I} + I} \right)}} = {{k_{op}\left( \frac{L}{L + K_{I}} \right)}^{n}\left( \frac{{\overset{\_}{K}}_{I}}{{\overset{\_}{K}}_{I} + I} \right)}} & (7)\end{matrix}$

For clarity of the discussion, only two mechanisms are described aboveto illustrate the principle of how different mechanistic models can bedifferentiated. In practice, different models such as competitivemechanism can be formulated and tested similarly. The method of modeldifferentiation described concerns only the measurement of rateconstants. In fact, the ratio of the current amplitude in the presenceand absence of an inhibitor is not only informative but also diagnostic.The amplitude ratio can be used to independently obtain affinityconstants for inhibitors and to verify them with those obtained fromrate constant measurements. Experimentally, the rate constant and themaximum current amplitude are collected from a single laser pulsephotolysis experiment and both are used. The methodology for determiningthe mechanism and efficacy of an inhibitor is based upon the measurementof the current from a sum of receptors (either in a whole cell or in apatch), rather than from a unitary conductance level as observed insingle channel recording. Therefore, the method described isparticularly useful to deal with a low conductance signal, which has sofar limited the study of the GluR2 channel by single channel recording.

The present invention relates to the discovery and characterization of agroup of water-soluble AMPA receptor inhibitors, which are RNA aptamersthat can fold into unique three-dimensional structures that confer highaffinity and specificity against biological targets. Using patch-clamprecording as a functional assay, we found that these aptamersreproducibly inhibited the glutamate-induced whole-cell current from thehuman embryonic kidney (HEK)-293 cells expressing the GluR2Q_(flip)receptor. The determination of the specific inhibition of the GluR2 AMPAreceptor by the selected aptamer using whole-cell recording of HEK-293cells expressing the receptor channel is shown in FIG. 2. FIG. 2Adepicts the whole-cell current response to 500 μM glutamate in theabsence and presence of 75 nM AN57. Inhibition by AN57 wasconcentration-dependent, as shown in FIG. 2B, with the K_(I,app) forAN57 determined to be 296±10 nM from the plot of the whole-cell currentin the absence, A and presence, A(I), of AN57 using Equation 1, yieldingthe intrinsic inhibition constant (K_(I)) of 133±5 nM calculated usingEqu. 8. The specific inhibition of the GluR2 channel by aptamer AN57 wasverified by using pool 2, the second-round library, or RN87, a differentsequence from AN57. Neither the library of randomized sequences nordifferent sequence showed any inhibition (FIG. 2C).

Next, we determined the minimized, yet fully functional, sequence.Guided by the secondary structure prediction using the Mfold program andby the activity assay using whole-cell recording, the sequence of theoriginal 98-nt aptamer was truncated from both ends (FIG. 2D) and thecorresponding versions were tested using whole-cell recording. Removalof 26 nucleotides (nt) from the 5′ direction (a constant region of thesequence template) resulted in a total loss of inhibitory function,suggesting that this sequence plays an essential role, likely astructural one, for the aptamer function. In contrast, progressivereduction of the sequence from the 3′ end showed that a 57 nt versionretained the activity whereas the 53 nt version did not (FIG. 2D). Thedeletion of 4 more nucleotides at the 3′ end of the 57-nt versionpresumably disrupted critical base paring, as predicted by Mfold. Thus,the 57-nt aptamer became the minimized working template for thesubsequent structural and functional studies.

In this successful selection of the aptamer, the GluR2 AMPA receptorsubunit was chosen to be the selection target because of its key role inmediating the calcium-dependent neuronal toxicity. Experimentally, GluR2was expressed in HEK-293 cells and the membrane that harbored thefunctional channel was used for SELEX. Thus, the aptamers selected wouldbe expected to recognize the total, functional GluR2 receptor.Furthermore, NBQX was chosen to selectively “elute” the aptamers thatwere bound to the NBQX site or to a mutually exclusive site(s) on thereceptor. The choice of NBQX was based on both its medical relevance andits known mechanism of action as a competitive inhibitor. Consequently,the selected aptamers would be predicted to be competitive inhibitors aswell. To confirm this, the dose response relationship of glutamate withthe GluR2Q_(flip) channel was measured in the absence and presence ofaptamer. As shown in FIG. 3A, the two curves converged at saturatingconcentrations of agonist, which is characteristic of competitiveinhibition, indicating that AN57 competitively inhibited the GluR2 AMPAreceptor. The effect of the aptamer on the dose-response curve isactually similar to the effect of NBQX, a known inhibitor (FIG. 6).Using a laser-pulse photolysis technique with caged glutamate, the rateof the receptor channel opening was measured (FIG. 3B). The observedrate constant of the GluR2Q_(flip) channel opening was slowed, asexpected, in the presence of aptamer. It should be further noted thatthe laser-pulse photolysis technique offered a ˜60 μs time resolution.Such time resolution was sufficient to enable us to measure the rate ofchannel opening before the receptor desensitization—a reaction thatleads to a glutamate-bound, but channel-closed, thus inactive, receptorstate. This technique therefore permits a critical screening ofaptamers.

The nanomolar affinity of aptamer AN57 (i.e., K_(I)=133 nM) comparesfavorably with the micromolar affinity values generally found for AMPAreceptor inhibitors prepared synthetically. However, as compared withNBQX, the “elution pressure” used in our SELEX, the affinity of theaptamer is >10-fold lower. At least two possibilities might explain thelower affinity of the aptamer. First, the sequence identified from theRNA library may not confer the most optimized secondary structure, andwe are attempting to select derivatives with higher affinity by librarydoping based on the existing sequence. Second, there might be multipleaptamer structures folded from the same sequence during the in vitrotranscription, and yet only one might be functional. If so, the K_(I) of133 nM would be an over-estimated value for the functional structure inthe mixture of all structures.

To investigate this possibility, we examined the in vitro RNAtranscripts on a native polyacrylamide gel (PAGE) (FIG. 4A). Two majorbands were observed, and termed as M1 and M2 (FIG. 4A, left panel). Thedifference in mobility in the native gel between M1 and M2 is indicativeof different structural features assumed from the same sequence, as onlya single sharp band was seen in denaturing gel (FIG. 4A, right panel).Furthermore, these two major structures were visible throughout thesequence reduction but were the clearest in the 57-nt aptamer (FIG. 4A).

Surprisingly, the functional test showed that neither M1 nor M2 alonewas inhibitory (FIG. 4B); but when combined, the inhibitory activity wasfully restored, as compared to the original, in vitro transcribed sample(FIG. 4B). Based on these results, we propose a simple model by which(FIG. 4C) binding of both M1 and M2 to their sites is sufficient tosterically block the entry of glutamate. Binding of either M1 or M2alone, however, is insufficient to set up the steric barrier to blockthe entrance of the glutamate binding site. Note that this model (FIG.4C) is also consistent with the competitive nature of the inhibition bythe aptamer (FIG. 3A).

To further test this hypothesis, we carried out a series of experiments(FIG. 4D). First, an equal molar mixture of the nonfunctional 53-nttranscript and the purified M2 yielded no inhibition, but the mixture ofthe 53-nt transcript and the purified M1 produced an inhibition as fullas the mixture of purified M1 and M2 (FIG. 4D). This finding suggeststhat the 53-nt sequence apparently can form an M2-like structure, butnot an M1-like structure. As a comparison, a nonfunctional,sequence-unrelated RNA, RN87, showed no inhibitory effect when mixedwith either M1 or M2 (FIG. 4D). These results support the model (FIG.4C) in which M1 and M2 bind to two separate sites, and further supportthe assumption that M1 and M2 are two structurally different species.Second, the results from the inhibition experiment using both agonistkainate (FIG. 7), instead of glutamate, and the GluR4_(flip) AMPAreceptor (FIG. 8), instead of GluR2Q_(flip), showed that the binding ofboth M1 and M2 as a collaborating pair was required for inhibition.These results were consistent with the model (FIG. 4C) by which theinhibition by the aptamer would be expected even with a differentagonist or with a different AMPA receptor. Knowing then that aptamerAN57 assumed two structures, M1 and M2, we further estimated theintrinsic inhibition constant or K_(I) of M1 by using 10-fold excess ofM2 or vice versa, according to Equ. 8. Under such a condition, K_(I) forM1 and M2 was calculated to be 63±10 and 66±18 nM respectively, usingEquation 9. These values suggest that both structures have nearly thesame affinity for the GluR2Q_(flip) receptor. Furthermore, theexperiment using different ratios of M1 and M2 revealed that thestoichiometry of the inhibition is 1:1. It should be noted, however,that the aptamer could alternatively bind deep into the glutamate siteto directly displace glutamate. Future structural investigations will beneeded to identify sites of binding for both M1 and M2, and todistinguish these models accordingly. These investigations are nowpossible because both the X-ray and NMR structures for the extracellularbinding domain of the GluR2Q_(flip) receptor channel have beendetermined, and both the agonist-bound and the competitiveantagonist-bound structures have been also resolved.

In one embodiment, the selection produces two structurally distinct RNAstructures encoded by a single sequence. Both theoretical andexperimental work has demonstrated the possibility that a single RNAsequence can assume multiple, distinctly folded structures. In thepresent instance, both structures are required to be present in a singlepreparation for a single function. Furthermore, these two structuresassumed by the same sequence are stable, as monitored in native PAGE,even after extreme treatments such as boiling at 100° C. in the presenceof 8 M urea, ethanol precipitation or freezing. Only after boiling at100° C. in the presence of 50% formamide for 15 min did M1 partiallyunfold into something else but not into M2. Under the same condition, M2remained essentially intact. The folding of these structures encoded bythe same sequence is at the present unclear. Whether the sequence ofAN57 assumes the structure of M1 or M2 is very likely determined bycertain events during the transcription process. It is unlikely thatthese structures are converted by a thermodynamic equilibrium. Thequestion of how these structures are formed is being pursued.

The stability of aptamer AN57 and its longer versions made it allpossible for the aptamer to survive through multiple rounds ofselections. For the same aptamer to be selected, the interaction betweenthe aptamer and the receptor must have been strong as well. A previousstudy of the electrostatic field of the extracellular binding domain ofthe GluR2 AMPA receptor with glutamate, a negatively charged molecule atphysiological pH, suggests that the surface of the receptor that makesup the entry of glutamate to its binding site is positively charged.Consequently this favorable electrostatic attraction is thought to steerglutamate to its site when a glutamate molecule approaches by freediffusion. As such, the negatively charged RNA molecules, like the onewe selected, could bind to the positively charged receptor surface. Sucha strong electrostatic attraction between the receptor and the stableRNA structures could be the basis of evolution through multiple roundsof selection, allowing the survival of such an aptamer from all otheralternative, competing structures.

The bi-structural feature of aptamer AN57 can be especially beneficialin developing more specific inhibitors/drugs against AMPA receptors thanNBQX, the classical competitive inhibitor that displaces agonist fromits binding site. This prediction is based on the novel mechanism ofaptamer inhibition in that the competitive inhibition is realized in asimultaneous recognition of the receptor by two collaboratingstructures. Thus, using this collaborating pair should provide a greaterspecificity, than using a single molecule, like NBQX. Consistent withthis prediction, AN57 was found to exhibit the improved specificitytowards the GluR4_(flip) channel, as compared to NBQX (FIG. 5A).Furthermore, AN57 showed no inhibition to the GluR6Q kainate receptor atthe concentration tested (FIG. 5A). This property is desirable becausekainate and AMPA receptor channels have different functionality in vivo.Therefore, as an initial proof of principle experiment, our resultssuggest the possibility of developing competitive inhibitors specific tosome AMPA receptor subunits, perhaps by using another AMPA receptorsubunit as the selection target.

Aptamer AN57 possesses additional superior inhibitory property over NBQXin that the aptamer retains the same inhibitory potency when the pHdrops from 7.4 to 6.4 (FIG. 5B) whereas NBQX loses its potencyby >3-fold. This acidic condition is clinically relevant to theinfarcted brain regions and thus the efficacy of inhibition by apotential drug at this acidic pH is a major requirement for an effectivestroke treatment. Thus, aptamer AN57 is a promising template forpotential drug development for the treatment of neurodegenerativeconditions such as post-stroke cellular lesion.

The aptamer we have generated represents a unique, water-solubletemplate for future design of better inhibitors with potentialtherapeutic values in the diagnosis and the treatment ofneurodegenerative disorders. The requirement of the simultaneous bindingof the two structurally distinct aptamer species to an AMPA receptor toexert competitive inhibition in a collaborating fashion can beconsidered advantageous over the conventional displacement model by asingle, competitive inhibitor. Such a use of this bi-structured aptamermay provide a higher regulatory ability as the lead compound in thedesign of subunit-specific inhibitors with nanomolar affinity for AMPAglutamate receptors.

Data Analysis

The inhibitory property of an RNA molecule was determined by the ratioof the glutamate-induced whole-cell current amplitude in the absence (A)and presence of the aptamer [A(I)]. A general scheme was formulated,below, in which binding of glutamate or ligand (L) to the unligandedreceptor form (R) leads to the opening of the channel ( RL₂ ). Bothsteps of binding were assumed to have the same equilibrium dissociationconstant (K₁). The number of ligand molecules bound to open an AMPAreceptor channel is generally considered to be two.

$\left. {\underset{\underset{\underset{\underset{RI}{\downharpoonleft \upharpoonright K_{1}}}{I}}{+}}{R} + {nL}}\overset{K_{1}}{\rightleftharpoons}{RL}_{n}\rightleftharpoons\overset{\_}{{RL}_{n}} \right.$

Shown in the same scheme is the competitive inhibition by an aptamer. Bythis mechanism, the apparent inhibition constant (K_(I, app)) can beobtained from the plot of A/A(I) versus the inhibitor concentration (I)using Equ. 8, and the intrinsic inhibition constant (K_(I)) can becalculated based on the K_(I,app) and the glutamate concentration (L)using Equ. 9:

$\begin{matrix}{\frac{A}{A(I)} = {1 + \frac{I}{K_{I,{app}}}}} & {{Equ}.\mspace{14mu} 8} \\{K_{I,{app}} = {K_{I}\left\lbrack {{\frac{1}{\Phi}\left( \frac{L}{K_{1}} \right)^{2}} + \left( \frac{L + K_{1}}{K_{1}} \right)^{2}} \right\rbrack}} & {{Equ}.\mspace{14mu} 9}\end{matrix}$

where Φ⁻¹ is the channel-opening equilibrium constant which wedetermined before. This scheme was used to estimate the apparent andintrinsic inhibition constants for aptamer AN57, assuming AN57 was asingle molecule. The same equation was also used to estimate theintrinsic inhibition constant for M1 and M2 individually. In this case,the inhibition constant for one structure was determined when the otherwas 10-fold in excess in its molar concentration. Because binding of onestructure did not elicit any current reduction, the single species-boundreceptor state (i.e., either RM1 or RM2 state) was considered to befunctionally equivalent to the R form (i.e., unliganded receptor form);only the RM1-M2 state was inhibitory.

Unless noted otherwise, each data point was an average of at least threemeasurements collected from at least three cells. Origin 7 (Origin Lab,Northampton, Mass.) was used for data analysis and plotting.Uncertainties reported refer to standard deviation from the mean.

The ultimate goal of identifying nucleic acid ligands such as thosedescribed herein is to develop selected aptamers as therapeutic anddiagnostic agents for the treatment of ALS and other excitotoxicdisorders.

Pharmaceutical Compositions

The invention, therefore, also includes pharmaceutical compositionscontaining nucleic acid aptamers of the invention. In some embodiments,the compositions are suitable for internal use and include an effectiveamount of a pharmacologically active compound of the invention, alone orin combination with one or more pharmaceutically acceptable carriers.The compounds are especially useful in that they have very goodsolubility.

Compositions of the invention can be used in a method for treating apatient or subject having a disease characterized by excessiveactivation of ionotropic glutamate receptors. Examples of diseasesamenable to treatment in accordance with the present invention includeamyotrophic lateral sclerosis (ALS), stroke, Alzheimer's disease andParkinson's syndrome. The method involves administering to the patientor subject a composition comprising a nucleic acid aptamer that bindsthe receptor involved with the pathology, so that binding of thecomposition to the target alters the biological function of the target,thereby treating the pathology.

The patient or subject to be treated by the methods of this inventioncan be a mammal, or more particularly, a human.

In practice, the compounds or their pharmaceutically acceptable salts,are administered in amounts which will be sufficient to exert theirdesired biological activity, e.g., inhibiting the binding of glutamateto its receptor. The preparation of pharmaceutical or pharmacologicalcompositions will be known to those of skill in the art in light of thepresent disclosure. Typically, such compositions may be prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injection;as tablets or other solids for oral administration; as time releasecapsules; or in any other form currently used, including eye drops,creams, lotions, salves, inhalants and the like. The use of sterileformulations, such as saline-based washes, by surgeons, physicians orhealth care workers to treat a particular area in the operating fieldmay also be particularly useful. Compositions may also be delivered viamicrodevice, microparticle or sponge.

Upon formulation, therapeutics will be administered in a mannercompatible with the dosage formulation, and in such amount as ispharmacologically effective. The formulations are easily administered ina variety of dosage forms, such as the type of injectable solutionsdescribed above, but drug release capsules and the like can also beemployed.

In this context, the quantity of active ingredient and volume ofcomposition to be administered depends on the host animal to be treated.Precise amounts of active compound required for administration depend onthe judgment of the practitioner and are peculiar to each individual.

The magnitude of a prophylactic or therapeutic dose of aptamer in theacute or chronic management of disease will vary with the severity andnature of the condition to be treated and the route of administration.The dose and perhaps the dose frequency will also vary according to theage, body weight and response of the individual patient. It may benecessary to use dosages outside the usual ranges in some cases, as willbe apparent to those skilled in the art. Further, it is noted that theclinician or treating physician will know how and when to interrupt,adjust, or terminate therapy in conjunction with individual patientresponse. The terms “a therapeutically effective amount” and “an amountsufficient to treat conditions characterized by excessive activation ofionotropic glutamate receptors are encompassed by the above-describeddosage amounts and dose frequency schedule.

Any suitable route of administration may be employed for providing thepatient with an effective dosage of the aptamer of the invention. Forexample, oral, rectal, parenteral (subcutaneous, intramuscular,intravenous), transdermal, aerosol and like forms of administration maybe employed. Dosage forms include tablets, troches, dispersions,suspensions, solutions, capsules, transdermal delivery systems, and thelike.

The pharmaceutical compositions of the present invention comprise anaptamer of the invention as the active ingredient, and may also containa pharmaceutically acceptable carrier, and optionally, other therapeuticingredients.

REFERENCES

-   1. Cluskey, S., and Ramsden, D. B. (2001) Mechanisms of    neurodegeneration in amyothrophic lateral sclerosis. Mol Pathol 54,    386-392.-   2. Charles, T., and Swash, M. (2001) Amyotrophic lateral sclerosis:    current understanding. J Neurosci Nurs 33, 245-253.-   3. Silani, V., Braga, M., Cardin, V., and Scarlato, G. (2001) The    pathogenesis of ALS: implications for treatment strategies. Neurol    Neurochir Pol 35, 25-39.-   4. Krampfl, K., Schlesinger, F., Wolfes, H., Dengler, R., and    Bufler, J. (2001) Functional diversity of recombinant human AMPA    type glutamate receptors: possible implications for selective    vulnerability of motor neurons. J Neurol Sci 191, 19-23.-   5. Iwasaki, Y., Ikeda, K., and Kinoshita, M. (1992) Plasma amino    acid levels in patients with amyotrophic lateral sclerosis. J Neurol    Sci 107, 219-222.-   6. Shaw, P. J., Forrest, V., Ince, P. G., Richardson, J. P., and    Wastell, H. J. (1995) CSF and plasma amino acid levels in motor    neuron disease: elevation of CSF glutamate in a subset of patients.    Neurodegeneration 4, 209-216.-   7. Lin, C. L., Bristol, L. A., Jin, L., Dykes-Hoberg, M., Crawford,    T., Clawson, L., and Rothstein, J. D. (1998) Aberrant RNA processing    in a neurodegenerative disease: the cause for absent EAAT2, a    glutamate transporter, in amyotrophic lateral sclerosis. Neuron 20,    589-602.-   8. Saroff, D., Delfs, J. Kuznetsov, D., and Geula, C. (2000)    Selective vulnerability of spinal cord motor neurons to non-NMDA    toxicity. Neuroreport 11, 1117-1121.-   9. Shaw, P. J., and Ince, P. G. (1997) Glutamate, excitotoxicity and    amyotrophic lateral sclerosis. J Neurol 244, S3-14.-   10. Urushitani, M., Nakamizo, T., Inoue, R., Sawada, H., Kihara, T.,    Honda, K., Akaike, A., and Shimohama, S. (2001) N-methyl-D-aspartate    receptor-mediated mitochondrial Ca(2+) overload in acute excitotoxic    motor neuron death: a mechanism distinct from chronic neurotoxicity    after Ca(2+) influx. J Neurosci Res 63, 377-387.-   11. Van Den Bosch, L., and Robberecht, W. (2000) Different receptors    mediate motor neuron death induced by short and long exposures to    excitotoxicity. Brain Res Bull 53, 383-388.-   12. Nakamura, R., Kamakura, K., Hirata, A., and Kwak, S. (1997)    Concentration-dependent changes in motor behavior produced by    continuous intrathecal infusion of excitatory amino acids in the rat    spinal cord. Brain Res Protoc 1, 385-390.-   13. Rothstein, J. D., Jin, L., Dykes-Hoberg, M., and    Kuncl, R. W. (1993) Chronic inhibition of glutamate uptake produces    a model of slow neurotoxicity. Proc Natl Acad Sci USA 90, 6591-6595.-   14. Brorson, J. R., Manzolillo, P. A., and Miller, R. J. (1994) Ca2+    entry via AMPA/KA receptors and excitotoxicity in cultured    cerebellar Purkinje cells. J Neurosci 14, 187-197.-   15. Turetsky, D. M., Canzoniero, L. M., Sensi, S. L., Weiss, J. H.,    Goldberg, M. P., and Choi, D. W. (1994) Cortical neurons exhibiting    kainite-activated Co2+ uptake are selectively vulnerable to    AMPA/kainite receptor-mediated toxicity. Neurobiol Dis 1, 101-110.-   16. Jonas, P., and Spruston, N. (1994) Mechanisms shaping    glutamate-mediated excitatory postsynaptic currents in the CNS. Curr    Opin Neurobiol 4, 366-372.-   17. Geiger, J. R., Melcher, T., Koh, D. S., Sakmann, B., Seeburg, P.    H., Jonas, P., and Monyer, H. (1995) Relative abundance of subunit    mRNAs determines gating and Ca2+ permeability of AMPA receptors in    principal neurons and interneurons in rat CNS. Neuron 15, 193-204.-   18. Williams, T. L., Ince, P. G., Oakley, A. E., and    Shaw, P. J. (1996) An immunocytochemical study of the distribution    of AMPA selective glutamate receptor subunits in the normal human    motor system. Neuroscience 74.185-198.-   19. Greig, A., Donevan, S. D., Mujtaba, T. J., Parks, T. N., and    Rao, M. S. (2000) Characterization of the AMPA-activated receptors    present on motoneurons. J Neurochem 74, 179-191.-   20. Vandenberghe, W., Robberecht, W., and Brorson, J. R. (2000) AMPA    receptor calcium permeability, GluR2 expression, and selective    motoneuron vulnerability. J Neurosci 20, 123-132.-   21. Dawson, D. A., Wadsworth, G., and Palmer, A. M. (2001) A    comparative assessment of the efficacy and side-effect liability of    neuroprotective compounds in experimental stroke. Brain Res 892,    344-350.-   22. Madsen, U., Stensbol, T. B., and Krogsgaard-Larsen, P. (2001)    Inhibitors of AMPA and kainite receptors. Curr Med Chem 8,    1291-1301.-   23. Nikam, S. S., and Kornberg, B. E. (2001) AMPA receptor    antagonists. Curr Med Chem 8, 155-170.-   24. Honore, T., Davies, S, N., Drejer, J., Fletcher, E. J.,    Jacobsen, P., Lodge, D., and Nielsen, F. E. (1998)    Quinoxalinediones: potent competitive non-NMDA glutamate receptor    antagonists. Science 241, 701-703.-   25. Sheardown, M. J., Nielsen, E. O., Hansen, A. J., Jacobsen, P.,    and Honore, T. (1990)    2,3-Dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline: a    neuroprotectant for cerebral ischemia. Science 247, 571-574.-   26. Abraham, G., Solyom, S., Csuzdi, E., Berzsenyi, P., Ling, I.,    Tarnawa, I., Hamori, T., Pallagi, I., Horvath, K., Andrasi, F.,    Kapus, G., Harsing, L. G., Jr., Kiraly, I., Patthy, M., and    Horvath, G. (2000) New non competitive AMPA antagonists. Bioorg Med    Chem 8, 2127-2143.-   27. Jackson, H., and Usherwood, P. N. (1988) Spider toxins as tools    for dissecting elements of excitatory amino acid transmission.    Trends Neurosci 11, 278-283.-   28. Iino, M., Koike, M., Isa, T., and Ozawa, S. (1996)    Voltage-dependent blockage of Ca(2+)-permeable AMPA receptors by    joro spider toxin in cultured rat hippocampal neurons. J Physiol    (Lond) 496, 431-437.-   29. Brackley, P., Goodnow, R., Jr., Nakanishi, K., Sudan, H. L., and    Usherwood, P. N. (1990) Spermine and philanthotoxin potentiate    excitatory amino acid responses of Xenopus oocytes injected with rat    and chick brain RNA. Neurosci Lett 114, 51-56.-   30. Dingledine, R., Borges, K., Bowie, D., and    Traynelis, S. F. (1999) The glutamate receptor ion channels.    Pharmacol Rev 51, 7-61.-   31. Hollmann, M., and Heinemann, S. (1994) Cloned glutamate    receptors. Annu Rev Neurosci 17, 31-108.-   32. Hollmann, M., Hartley, M., and Heinemann, S. (1991) Ca2+    permeability of KA-AMPA-gated glutamate receptor channels depends on    subunit composition. Science 252, 851-853.-   33. Seeburg, P. H. (1993) The TINS/TiPS Lecture, The molecular    biology of mammalian glutamate receptor channels. Trends neurosci    16, 359-365.-   34. Nakanishi, N., Shneider, N. A., and Axel, R. (1990) A family of    glutamate receptor genes: evidence for the formation of    heteromultimeric receptors with distinct channel properties. Neuron    5, 569-581.-   35. Collingridge, G. L., and Lester, R. A. (1989) Excitatory amino    acid receptors in the vertebrate central nervous system. Pharmacol    Rev 41, 143-210.-   36. Wo, Z. G., and Oswald, R. E. (1995) Unraveling the modular    design of glutamate-gated ion channels. Trends Neurosci 18, 161-168.-   37. Hollmann, M., Maron, C., and Heinemann, S. (1994)    N-glycosylation site tagging suggests a three transmembrane domain    topology for the glutamate receptor GluR1. Neuron 13, 1331-1343.-   38. Bennett, J. A., and Dingledine, R. (1995) Topology profile for a    glutamate receptor: three transmembrane domains and a channel-lining    reentrant membrane loop. Neuron 14, 373-384.-   39. Armstrong, N., Sun, Y., Chen, G. Q., and Gouaux, E. (1998)    Structure of a glutamate-receptor ligand-binding core in complex    with kainite. nature 395, 913-917.-   40. Dev, K. K., and Henley, J. M. (1998) The regulation of AMPA    receptor-binding sites. Mol Neurobiol 17, 33-58.-   41. Trist, D. G. (2000) Excitatory amino acid agonists and    antagonists: pharmacology and therapeutic applications. Pharm Acta    Helv 74, 221-229.-   42. Turski, L., Huth, A., Sheardown, M., McDonald, F., Neuhaus, R.,    Schneider, H. H., Dirnagl, U., Wiegand, F., Jacobsen, P., and    Ottow, E. (1998) ZK200775: a phosphonate quinoxalinedione AMPA    antagonist for neuroprotection in stroke and trauma. Proc Natl Acad    Sci USA 95, 10960-10965.-   43. Swanson, G. T., Kamboj, S. K., and Cull-Candy, S. G. (1997)    Single-channel properties of recombinant AMPA receptors depend on    RNA editing, splice variation, and subunit composition. J Neurosci    17, 58-69.-   44. White, R. R., Sullenger, B. A., and Rusconi, C. P. (2000)    Developing aptamers into therapeutics [In Process Citation]. J Clin    Invest 106, 929-934.-   45. Brody, E. N., and Gold, L. (2000) Aptamers as therapeutic and    diagnostic agents [In Process Citation]. J Biotechnol 74, 5-13.-   46. Hesselberth, J., Robertson, M. P., Jhaveri, S., and    Ellington, A. D. (2000) In vitro selection of nucleic acids for    diagnostic applications [In Process Citation]. J Biotechnol 74,    15-25.-   47. Brody, E. N., Willis, M. C., Smith, J. D., Jayasena, S., Zichi,    D., and Gold, L. (1999) The use of aptamers in large arrays for    molecular diagnostics. Mol Diagn 4, 381-388.-   48. Ellington, A. D., and Szostak, J. W. (1990) In vitro selection    of RNA molecules that bind specific ligands. Nature 346, 818-822.-   49. Tuerk, C., and Gold, L. (1990) Systematic evolution of ligands    by exponential enrichment: RNA ligands to bacteriophage T4 DNA    polymerase. Science 249, 505-510.-   50. Jhaveri, S., Rajendran, M., and Ellington, A. D. (2000) In vitro    selection of signaling aptamers. Nat Biotechnol 18, 1293-1297.-   51. Stojanovic, M. N., de Prada, P., and Landry, D. W. (2001)    Aptamer-based folding fluorescent sensor for cocaine. J Am Chem Soc    123, 4928-4931.-   52. Ruckman, J., Green, L. S., Beeson, J., Waugh, S., Gillette, W.    L., Henninger, D. D., Claesson-Welsh, L., and Janjic, N. (1998)    2′-Fluoropyrimidine RNA-based aptamers to the 165-amino acid form of    vascular endothelial growth factor (VEGF165). Inhibition of receptor    binding and VEGF-induced vascular permeability through interactions    requiring the exon 7-encoded domain. J Biol Chem 273, 20556-20567.-   53. Sun, S. (2000) Technology evaluation: SELEX, Gilead Sciences    Inc. Curr Opin Mol Ther 2, 100-105.-   54. Hess, G. P., and Grewer, C. (1998) Development and application    of caged ligands for neurotransmitter receptors in transient kinetic    and neuronal circuit mapping studies. Methods Enzymol 291, 443-473.-   55. Niu, L., and Hess, G. P. (1993) An acetylcholine receptor    regulatory site in BC3H1 cells: characterized by laser-pulse    photolysis in the microsecond-to-millisecond time region.    Biochemistry 32, 3831-3835.-   56. Niu, L., Abood, L. G., and Hess, G. P. (1995) Cocaine: mechanism    of inhibition of a muscle acetylcholine receptor studied by a    laser-pulse photolysis technique. Proc Natl Acad Sci USA 92,    12008-12012.-   57. Hess, G. P. (1993) Determination of the chemical mechanism of    neurotransmitter receptor-mediated reactions by rapid chemical    kinetic techniques. Biochemistry 32, 989-1000.-   58. Kaplan, J. H. (1990) Photochemical manipulation of divalent    cation levels. Annu Rev Physiol 52, 897-914.-   59. McCray, J. A., and Trentham, D. R. (1989) Properties and uses of    photoreactive caged compounds. Annu Rev Biophys Chem 18, 239-270.-   60. Matsubara, N., Billington, A. P., and Hess, G. P. (1992) How    fast does an acetylcholine receptor channel open? Laser-pulse    photolysis of an inactive precursor of carbamoylcholine in the    microsecond time region with BC3H1 cells. Biochemistry 31,    5507-5514.-   61. Wieboldt, R., Gee, K. R., Niu, L., Ramesh, D., Carpenter, B. K.,    and Hess, G. P. (1994) Photolabile precursors of glutamate:    synthesis, photochemical properties, and activation of glutamate    receptors on a microsecond time scale. Proc Natl Acad Sci USA 91,    8752-8756.-   62. Gee, K. R., Niu, L., Schaper, K., Jayaraman, V., and    Hess, G. P. (1999) Synthesis and photochemistry of a photolabile    precursor of N-methyl-D-asparate (NMDA) that is photolyzed in the    microsecond time region and is suitable for chemical kinetic    investigations of the NMDA receptor. Biochemistry 38, 3140-3147.-   63. Niu, L., Gee, K. R., Schaper, K., and Hess, G. P. (1996)    Synthesis and photochemical properties of a kainate precursor and    activation of kainate and AMPA receptor channels on a microsecond    time scale. Biochemistry 35, 2030-2036.-   64. Hess, G. P. Ulrich, H., Breitinger, H. G., Niu, L., Gameiro, A.    M., Grewer, C., Srivastava, S., Ippolito, J. E., Lee, S. M.,    Jayaraman, V., and Coombs, S. E. (2000) Mechanism-based discovery of    ligands that counteract inhibition of the nicotinic acetylcholine    receptor by cocaine and MK-801. Proc Natl Acad Sci USA 97,    13895-13900.-   65. Ulrich, H., Ippolito, J. E., Pagan, 0. R., Eterovic, V. A.,    Hann, R. M., Shi, H., L is, J. T., Eldefrawi, M. E., and    Hess, G. P. (1998) In vitro selection of RNA molecules that displace    cocaine from the membrane-bound nicotinic acetylcholine receptor.    Proc Natl Acad Sci USA 95, 14051-14056.-   66. Bruel, C., Cha, K., Niu, L., Reeves, P. J., and    Khorana, H. G. (2000) Rhodopsin kinase: two mAbs binding near the    carboxyl terminus cause time-dependent inactivation. Proc Natl Acad    Sci USA 97, 3010-3015.-   67. Shi, H., Hoffman, B. E., and L is, T. J. (1997) A specific RNA    hairpin loop structure binds the RNA recognition motifs of the    Drosophila SR protein B52. Mol Cell Biol 17, 2649-2657.-   68. Camu, W., and Henderson, C. E. (1992) Purification of embryonic    rat motoneurons by panning on a monoclonal antibody to the    low-affinity NGF receptor. J Neurosci Methods 44, 59-70.-   69. Camu, W., and Henderson, C. E. (1994) Rapid purification of    embryonic rat motoneurons: an in vitro model for studying MND/ALS    pathogenesis. J Neurol Sci 124, 73-74.-   70. Washburn, M. S., and Dingledine, R. (1996) Block of    alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)    receptors by polyamines and polyamine toxins. J Pharmacol Exp Ther    278, 669-678.-   71. Gold, L. (1995) Oligonucleotides as research, diagnostic, and    therapeutic agents. J Biol Chem 270, 13581-13584.-   72. Chen, C., and Okayama, H. (1987) High-efficiency transformation    of mammalian cells by plasmid DNA. Mol Cell Biol 7, 2745-2752.-   73. Hamill, O. P., Marty, A., Neher, E., Sakmann, B., and    Sigworth, F. J. (1981) Improved patch-clamp techniques for    high-resolution current recording from cells and cell-free membrane    patches. Pflugers Arch 391, 85-100.-   74. Udgaonkar, J. B., and Hess, G. P. (1987) Chemical kinetic    measurements of a mammalian acetylcholine receptor by a    fast-reaction technique. Proc Natl Acad Sci USA 84, 8758-8762.-   75. L. I. Bruijn, T. M. Miller, D. W. Cleveland, Annu Rev Neurosci    27, 723 (2004).-   76. S. Solyom, I. Tarnawa, Curr Pharm Des 8, 913 (2002).-   77. K. Umemura et al., J Clin Pharmacol 37, 719 (1997).-   78. G. Li, W. Pei, L. Niu, Biochemistry 42, 12358 (2003).-   79. G. Li, Z. Sheng, Z. Huang, L. Niu, Biochemistry 44, 5835 (2005).-   80. L. Nowak, P. Bregestovski, P. Ascher, A. Herbet, A. Prochiantz,    Nature 307, 462 (1984).-   81. P. R. Heath, P. J. Shaw, Muscle Nerve 26, 438 (2002).-   82. P. Van Damme, M. Leyssen, G. Callewaert, W. Robberecht, L. Van    Den Bosch, Neurosci Lett 343, 81 (2003).-   83. T. Weiser, Curr Drug Targets CNS Neurol Disord 4, 153 (2005).-   84. M. Shimizu-Sasamata et al., J Pharmacol Exp Ther 276, 84 (1996).-   85. X. Fan, H. Shi, K. Adelman, J. T. Lis, Proc Natl Acad Sci USA    101, 6934 (2004).-   86. H. Shi, B. E. Hoffman, J. T. Lis, Proc Natl Acad Sci USA 96,    10033 (1999).-   87. R. R. Breaker, Nature 432, 838 (2004).-   88. M. Zuker, Nucleic Acids Res 31, 3406 (2003).-   89. G. Li, R. E. Oswald, L. Niu, Biochemistry 42, 12367 (2003).-   90. R. Knight, M. Yarus, Nucleic Acids Res 31, e30. (2003).-   91. N. Armstrong, E. Gouaux, Neuron 28, 165 (2000).-   92. R. L. McFeeters, R. E. Oswald, Biochemistry 41, 10472 (2002).-   93. M. A. Huynen, P. F. Stadler, W. Fontana, Proc Natl Acad Sci USA    93, 397 (1996).-   94. E. A. Schultes, D. P. Bartel, Science 289, 448 (2000).-   95. M. Kubo, E. Ito, Proteins 56, 411 (2004).-   96 J. Lerma, A. V. Paternain, A. Rodriguez-Moreno, J. C.    Lopez-Garcia, Physiol Rev 81, 971 (2001).-   97. G. Li, W. Pei, L. Niu, Biochemistry 42, 12358 (2003).-   98. C. Chen, H. Okayama, Mol Cell Biol 7, 2745 (1987).-   99. Z. Huang, G. Li, W. Pei, L. A. Sosa, L. Niu, J Neurosci Methods    142, 159 (2005).-   100. O. P. Hamill, A. Marty, E. Neher, B. Sakmann, F. J. Sigworth,    Pflugers Arch 391, 85 (1981).-   101. J. B. Udgaonkar, G. P. Hess, Proc Natl Acad Sci USA 84, 8758    (1987).

The invention claimed is:
 1. An isolated nucleic acid comprising thenucleotide sequence of at least nucleotides 1-57 of SEQ ID NO: 5 andbetween 1 to 40 additional contiguous nucleotides corresponding tonucleotides 58 to 97 of SEQ ID NO: 5, wherein said isolated nucleic acidbinds to a glutamate receptor.
 2. The isolated nucleic acid of claim 1,wherein said nucleic acid is RNA.
 3. The isolated nucleic acid of claim1, wherein the nucleic acid contains between 58 and 150 nucleotides. 4.The isolated nucleic acid of claim 1, wherein the nucleic acid containsbetween 58 and 100 nucleotides.
 5. The isolated nucleic acid of claim 1,wherein said nucleic acid contains one or more chemically modifiednucleotides.
 6. The isolated nucleic acid of claim 5 wherein the one ormore chemically modified nucleotides has a 2′ fluoro substituent.
 7. Theisolated nucleic acid of claim 1, wherein said nucleic acid inhibitsglutamate receptor function.
 8. The isolated nucleic acid of claim 1wherein the glutamate receptor is of theα-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) subtype.
 9. Anisolated DNA that codes for the RNA of claim
 2. 10. A method ofinhibiting a glutamate receptor comprising contacting said receptor withthe nucleic acid of claim
 1. 11. The method of claim 10, wherein theglutamate receptor is of the AMPA subtype.